title: Posttranslational modification of CENP-A influences the conformation of centromeric chromatin.
abstract: Centromeres are chromosomal loci required for accurate segregation of sister chromatids during mitosis. The location of the centromere on the chromosome is not dependent on DNA sequence, but rather it is epigenetically specified by the histone H3 variant centromere protein A (CENP-A). The N-terminal tail of CENP-A is highly divergent from other H3 variants. Canonical histone N termini are hotspots of conserved posttranslational modification; however, no broadly conserved modifications of the vertebrate CENP-A tail have been previously observed. Here, we report three posttranslational modifications on human CENP-A N termini using high-resolution MS: trimethylation of Gly1 and phosphorylation of Ser16 and Ser18. Our results demonstrate that CENP-A is subjected to constitutive initiating methionine removal, similar to other H3 variants. The nascent N-terminal residue Gly1 becomes trimethylated on the α-amino group. We demonstrate that the N-terminal RCC1 methyltransferase is capable of modifying the CENP-A N terminus. Methylation occurs in the prenucleosomal form and marks the majority of CENP-A nucleosomes. Serine 16 and 18 become phosphorylated in prenucleosomal CENP-A and are phosphorylated on asynchronous and mitotic nucleosomal CENP-A and are important for chromosome segregation during mitosis. The double phosphorylation motif forms a salt-bridged secondary structure and causes CENP-A N-terminal tails to form intramolecular associations. Analytical ultracentrifugation of phospho-mimetic CENP-A nucleosome arrays demonstrates that phosphorylation results in greater intranucleosome associations and counteracts the hyperoligomerized state exhibited by unmodified CENP-A nucleosome arrays. Our studies have revealed that the major modifications on the N-terminal tail of CENP-A alter the physical properties of the chromatin fiber at the centromere.
authors.affiliation: Department of Cell Biology, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Aaron O
authors.initials: AO
authors.lastname: Bailey
authors.forename: Tanya
authors.initials: T
authors.lastname: Panchenko
authors.forename: Kizhakke M
authors.initials: KM
authors.lastname: Sathyan
authors.forename: Janusz J
authors.initials: JJ
authors.lastname: Petkowski
authors.forename: Pei-Jing
authors.initials: PJ
authors.lastname: Pai
authors.forename: Dina L
authors.initials: DL
authors.lastname: Bai
authors.forename: David H
authors.initials: DH
authors.lastname: Russell
authors.forename: Ian G
authors.initials: IG
authors.lastname: Macara
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Shabanowitz
authors.forename: Donald F
authors.initials: DF
authors.lastname: Hunt
authors.forename: Ben E
authors.initials: BE
authors.lastname: Black
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Foltz
mesh_terms: D001324:Autoantigens / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D002503:Centromere / Q000737:chemistry*; D000076247:Centromere Protein A; D002843:Chromatin / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D044127:Epigenesis, Genetic / Q000235:genetics*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008745:Methylation; D008968:Molecular Conformation; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D014461:Ultracentrifugation
chemical_list: D001324:Autoantigens; C000619700:CENPA protein, human; D018797:Cell Cycle Proteins; D000076247:Centromere Protein A; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; C062521:RCC1 protein, human
keywords: epigenetics; kinetochore; mass spectrometry